Journal: PLoS ONE

Article Title: Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

doi: 10.1371/journal.pone.0093335

Figure Lengend Snippet: Effect of p53 inhibitor (PFT-α) on (A) the expression of apoptotic genes as determined by immunoblot analysis and (B) cell death in conjugate treated LNCaP cells as determined by MTT assay. Data are the mean ± SEM of three independent experiments. # and *indicates significant difference with respect to the controls for p53 and caspsase-3 proteins respectively at p <0.05. (C) Immunoblot analysis to show the phosphorylation patterns of Akt and ERK in PC-3 and (D) LNCaP cells in response to varying doses of conjugate and resveratrol treatments. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *represents statistically significant difference with respect to control for each group at p <0.05.

Article Snippet: Pifithrin-α (p53 inhibitor), antibodies for caspase-3, Bax, Akt, p-Akt, ERK, p-ERK, SRC-1, GRIP-1, N-CoR, β-actin and small interfering RNAs (siRNAs) against Akt (sc-43609), ERK (sc-35335) and control (sc-37007; negative control for experiments using targeted siRNA transfection; each consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, MTT Assay

Journal: PLoS ONE

Article Title: Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

doi: 10.1371/journal.pone.0093335

Figure Lengend Snippet: Effects of siRNA mediated silencing of Akt and ERK on conjugate-induced apoptosis of (A) PC-3 and (B) LNCaP cells. LNCaP and PC-3 PCa cells were transfected with siRNAs (at a final concentration of 100 nM) using polyfect transfection reagent. After 24 h of transfection the cells were treated with 20 μM conjugate and allowed to grow for another 24 h. The cell lysates were prepared and the level of p-Akt, p-ERK and cleaved caspase-3 proteins were detected by immunoblot analysis. The histogram on the right panel of each figure represents densitometric analyses of the image data and expressed as percent of control where the results are mean ± SEM of three independent experiments. *and # represents statistically significant difference with respect to control and 20 μM conjugate treated groups respectively at p <0.05.

Article Snippet: Pifithrin-α (p53 inhibitor), antibodies for caspase-3, Bax, Akt, p-Akt, ERK, p-ERK, SRC-1, GRIP-1, N-CoR, β-actin and small interfering RNAs (siRNAs) against Akt (sc-43609), ERK (sc-35335) and control (sc-37007; negative control for experiments using targeted siRNA transfection; each consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Concentration Assay, Western Blot

Journal: PLoS ONE

Article Title: Pterostilbene-Isothiocyanate Conjugate Suppresses Growth of Prostate Cancer Cells Irrespective of Androgen Receptor Status

doi: 10.1371/journal.pone.0093335

Figure Lengend Snippet: Conjugate inhibits both androgen receptor and ERK signalling and finally contributing to its downstream effects of decreased cell viability and increased apoptosis in androgen receptor positive LNCaP cells. In case of androgen receptor negative PC-3 cells, inhibition of Akt and its downstream targets contributes to conjugate mediated decrease in cell viability and increased apoptosis.

Article Snippet: Pifithrin-α (p53 inhibitor), antibodies for caspase-3, Bax, Akt, p-Akt, ERK, p-ERK, SRC-1, GRIP-1, N-CoR, β-actin and small interfering RNAs (siRNAs) against Akt (sc-43609), ERK (sc-35335) and control (sc-37007; negative control for experiments using targeted siRNA transfection; each consists of a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Metastasis of Tumor Cells Is Enhanced by Downregulation of Bit1

doi: 10.1371/journal.pone.0023840

Figure Lengend Snippet: A and B. Exponentially growing stable controlshRNA and Bit1shRNA knockdown pools derived from MCF7(A) and B16F1(B) were lysed, and the total lysate was subjected to immunoblotting to detect the phosphorylated Erk (pErk), total Erk(tErk), active Mek (pMek), total Mek (tMek), Bit1, and β-actin. C and D. Total cell lysates from stable MCF7controlshRNA and Bit1shRNA knockdown pools were subjected to an Erk phosphatase assay as described in . A representative immunoblot of isolated His-6-tagged Erk2 is shown to reveal pErk2 or total Erk2 levels (C). The relative intensity of pErk2/tErk was determined using NIH Image J software, and the values represent the average of at least three independent experiments (D). E, F and G. Stable Hela control clone#1 and Bit1RNAi#21 clone were transfected with control- or Erk2-specific siRNAs; 48 h post-transfection, cells were harvested and subjected to immunoblotting (E) with antibodies against total Erk2 and phosphorylated Erk1/2 (pErks). In parallel, cells were subjected to a fibronectin cell adhesion (F) and QCM boyden chamber migration assays (G) as described in . In D, F and G, results are representative of three independent experiments, *p<0.05 (Student's t test).

Article Snippet: The control and ERK2 specific siRNAs were purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Western Blot, Phosphatase Assay, Isolation, Software, Transfection, Migration

Journal: Cell reports

Article Title: The ribosomal RNA processing 1B:protein phosphatase 1 holoenzyme reveals non-canonical PP1 interaction motifs

doi: 10.1016/j.celrep.2022.111726

Figure Lengend Snippet:

Article Snippet: pEGFP(C1)-PP1α , Addgene , ID 44224.

Techniques: Virus, Expressing, Recombinant, Mutagenesis, Clone Assay, Protease Inhibitor, Modification, Bicinchoninic Acid Protein Assay, Software

Journal: bioRxiv

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1101/2022.08.09.503353

Figure Lengend Snippet: (A) A schematic representation of in vitro biochemical analyses used in this study. Grey and white circles without labels, proteins; a black curved line, a nascent RNA molecule. (B) Left, silver-stained recombinant ERK1 (K1) and ERK2 (K2) proteins used in in vitro analyses. SM, standard protein size marker. Right, the result of immunoblotting confirming ERK1 and ERK2. (C) Immobilized template assay results showing the increased occupancies of Pol II, CDK9, and MED23 by ERK2 on the EGR1 TSS. Error bars, standard deviation throughout the figures. *P < 0.05, ***P < 0.005, not significant (ns). (D) SDS-PAGE and silver-staining of the constitutively active ERK1m and ERK2m proteins. (E) Immobilized template assay results of Pol II, CDK9, and MED23. (F) in vitro transcription assay results showing EGR1 gene activation by ERK2 and ERK2m. **P < 0.01, ***P < 0.005, ****P < 0.0005.

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: In Vitro, Staining, Recombinant, Marker, Western Blot, Standard Deviation, SDS Page, Silver Staining, Transcription Assay, Activation Assay

Journal: bioRxiv

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1101/2022.08.09.503353

Figure Lengend Snippet: (A) Immobilized template assay results presenting TOP2B enrichment by ERK1m and ERK2 on the WT template but not on the ELK1 m template. (B) Deubiquitinated TOP2B used for the in vitro kinase assays, silver-stained. (C) Silver-stained reactions following the in vitro kinase assay. (D) Autoradiograms of the in vitro kinase assay with TOP2B and ERKs. ELK1, a positive control. (E) In vitro kinase assay followed by SDS-PAGE for the preparation of mass spectrometry analyses. Red boxes showing sliced gel pieces used for the analyses. MS, mass spectrometry. (F) A flow chart showing the steps of mass spectrometry analyses performed in this study. (G) A schematic representation of the TOP2B residues phosphorylated by ERK1m (green), ERK2m (red), or both kinases (blue).

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: In Vitro, Staining, Kinase Assay, Positive Control, SDS Page, Mass Spectrometry

Journal: bioRxiv

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1101/2022.08.09.503353

Figure Lengend Snippet: (A) qRT-PCR results presenting the effects of ERK2 catalytic inhibition on EGR1 and HSP70 transcription. HEK293 cells were not synchronized. ***P < 0.005. (B) qRT-PCR results presenting the effects of ERK2 catalytic inhibition on the EGR 1 and FOS transcription. HEK293 cells were synchronized at G0 (S0) before they were serum-induced to early G1 (S15). **P < 0.01, ***P < 0.005, ****P < 0.0005. (C) ChIP-qPCR results showing S2 Pol II occupancy changes in the EGR1 gene with or without functional ERK2. ****P < 0.0005. (D) ChIP-qPCR results showing a dramatic increase of TOP2B at EGR1 upon the catalytic inhibition of ERK2. *P < 0.05, ***P < 0.005, ****P < 0.0005. (E) Immunoblots of ERK1 (K1 KD ), ERK2 (K2 KD ), and ERK1/ERK2 double KD (Double KD ) HEK293 cells. Tubulin, a reference. (F) qRT-PCR and ChIP-qPCR results presenting the effects of ERK KDs on EGR1 transcription (left) and TOP2B occupancies at the EGR1 TSS (right), respectively. *P < 0.05. (G) qRT-PCR and ChIP-qPCR results presenting the effects of ERK KDs on FOS transcription (left) and TOP2B occupancies at the FOS TSS (right), respectively. *P < 0.05, ***P < 0.005.

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: Quantitative RT-PCR, Inhibition, Functional Assay, Western Blot

Journal: Communications Biology

Article Title: BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer

doi: 10.1038/s42003-020-01263-y

Figure Lengend Snippet: In SNU1235, SNU1047, HT29 and LS180 cell lines, BRAF, MEK, ERK1 and ERK2 were knocked down by transient transfection of RNA interference for 24 h, alone ( a ) or in combination with a dose fixed of D ( b ). Molecular effects on protein expression was analyzed by Western Blot (WB) using specific antibodies (β-Actin and α tubulin are shown as protein loading and blotting control). IL-8 was measured after 24 h of culture in serum-free medium, using IL-8 ELISA; IL-8 levels were measured as pg/mL and results are expressed as % of untreated control levels. Results of a representative experiment out of three independent experiments performed ( a ) or the average of three independent experiments ( b ) are shown. p-values indicate statistically significant differences ( p < 0.05 by 2-tailed Student’s t test) for the comparison between treated/silenced and non-treated/silenced conditions of growth.

Article Snippet: Cells were transfected with either a siRNA against BRAF (sequence: 5′-UACACCAGCAAGCUAGAUGCA-3′), MEK and ERK1 (Santa Cruz Biotechnology, Santa Cruz, CA) or shRNA plasmids against ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay